Coagulation proteases are trypsin-like enzymes with similar three-dimensional structures, unlike trypsin; however, they exhibit a high degree of specificity, assemble on membrane surfaces in the presence of calcium and require cofactors to function. Cofactors bind to coagulation proteases and/or their substrates to improve the rate of catalytic reactions by several orders of magnitude. Recent structural, kinetic and mutagenesis data suggest that such dramatic improvement in the rate of catalytic reactions may primarily be mediated by cofactor-dependent exosite interactions between these proteins. The high rates of factor Va-dependent prothrombin activation by factor Xa; thrombomodulin-dependent protein C activation by thrombin; tissue-factor dependent factors IX and X activation by factor Vila; and factor Villa-dependent factor X activation by factor IXa, have all been attributed to such interactions. The molecular basis for recognition specificity of these cofactor/enzyme/substrate interactions is not well understood. We hypothesize that a limited number of key divergent residues on the conserved homologous surface loops of coagulation proteases and/or their zymogens provide recognition sites, termed "exosites," for these specific interactions. The overall objective of this project is to identify and map these specific macromolecular interaction sites on coagulation factors. We propose to 1) use crystal structures, molecular models, mutagenesis and kinetic approaches to identify candidate residues on homologous surface loops at the same 3-dimensional locations on coagulation proteases and/or their zymogens that might be involved in determination of specificity; and 2) use crystal structures and molecular models to design coagulation factors that may shed light on the mechanism by which coagulation cofactors function.